DESCRIPTION: This application proposes experiments to investigate the molecular basis of patterning in the early Drosophila embryo. Dr. Small will focus his attention on the expression of the pair-rule gene, even-skipped (eve). This gene is expressed in a pattern of seven stripes, each 5-6 cells wide, in the cellularizing Drosophila embryo; eve expression is controlled by the products of maternal effect and gap genes, including bicoid (bcd), hunchback (hb), giant (gt) and Kruppel (Kr). Each of these genes encodes a DNA-binding protein. However, other, as yet unidentified proteins are also likely to be involved in the regulation of eve expression. Dr. Small will concentrate his analysis on the second most anterior stripe of eve expression. Transcription of eve in this region of the embryo is controlled by a 480 bp enhancer located upstream of the eve coding region. The enhancer contains binding sites for each of the four proteins known to regulate eve expression. Dr. Small's project is, in essence, a detailed analysis of the function of this enhancer. He will attempt to determine if activation of eve involves cooperative binding of the Bcd and Hb proteins to sites within the enhancer, and if repression involves either competitive binding of Gt and Kr proteins to DNA, or inhibitory protein-protein interactions. Dr. Small will also attempt to identify new protein binding sites within the stripe 2 enhancer, and to assess their functional significance in vivo by site- directed mutagenesis and P element transformation. Another aim is to use mutagenesis and P element transformation to determine the significance of the number, affinity, arrangement and spacing of the various binding sites within the stripe 2 enhancer. Dr. Small will also investigate the nature of the sequences that ensure the functional autonomy of the stripe 2 enhancer. The preceding aims have been carried forward from the previous submissions, albeit with some modifications. A new aim in this proposal is to test whether the gap gene proteins function as gradient morphogens in determining the domain of eve expression. Dr. Small proposes two approaches. First, he will systematically change the dosage of several gap genes and assess the effect on eve expression. Second, he will misexpress gap genes from a promoter under the control of the eve stripe 2 enhancer and assess the effect on eve expression. The latter approach is based on pilot experiments in which the gap gene knirps (kni), driven by the eve stripe 2 enhancer, was successfully misexpressed in the developing blastoderm. The results of these experiments indicate that knirps protein functions as a gradient morphogen, i.e., its ability to repress pair rule gene expression in a particular region of the embryo depends on its concentration in that region. A concentration either too low or too high precludes repressor function. Dr. Small proposes to extend this method to assess the effects of misexpressing the gap genes giant and Kruppel in the developing embryo. He hypothesizes that they, too, are gradient morphogens.